The phosphorylation of the beta-carboxyl group of aspartic acid is the commitment step toward the biosynthesis of four of the twenty common amino acids. In many organisms the amino acids lysine, threonine, methionine and isoleucine each derive a portion of their carbon skeleton from aspartic acid. This biosynthetic pathway is coming under increasing scrutiny both in microorganisms and plants, with the aim of identifying lead compounds that can serve as models for the synthesis of potential drugs or herbicides against target enzymes in this pathway. Progress in this area will be supported by, and its ultimate success will depend on, a detailed knowledge of the mechanisms of these enzyme-catalyzed transformations. The goal of this research project is to compare a group of three functionally related enzymes in Escherichia coli that each catalyze this phosphorylation of aspartic acid. Each of these enzymes is differentially regulated at the genetic and/or at the metabolic level, and two of these enzymes are bifunctional. However, sequence and homology comparison among the aspartokinases have shown that this family of enzymes was derived from a common ancestral gene. Several specific questions will be addressed by this research proposal: (1) Do each of these enzymes catalyze this phosphorylation by the same mechanism?; (2) Can these aspartokinases be distinguished based on their substrate specificity of their regiospecificity?; and (3) What are the structural relationships among these isofunctional enzymes? Each of these aspartokinases will be purified, by using selective dye ligand chromatography, from genetically engineered strains of E. coli that carry the individual genes on high copy plasmids. The kinetic mechanisms of these enzymes will be examined by a methodology that includes initial velocity, product, and dead-end inhibition studies. The identification of potentially important amino acid residues at the catalytic and allosteric sites of these enzymes will be determined by the combined application of pH dependent kinetic studies and chemical modification and protection studies. This set of identified amino acid residues will be narrowed by sequence comparisons to produce a list of target amino acids for oligonucleotide-directed mutagenesis studies. Genetic engineering techniques will also be used to separately express each of the distinct structural domains in the aspartokinases. The formation of hybrid subunits from the domains obtained from the different isofunctional enzymes will permit an examination of the communication between the various domains that leads to each active bifunctional, allosteric enzyme.